scholarly journals β-Sitosterol 3-O-D-glucoside increases ceramide levels in the stratum corneum via the up-regulated expression of ceramide synthase-3 and glucosylceramide synthase in a reconstructed human epidermal keratinization model

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248150
Author(s):  
Shogo Takeda ◽  
Shuko Terazawa ◽  
Hiroshi Shimoda ◽  
Genji Imokawa
Keyword(s):  
Stratum Corneum ◽  
Mrna Expression ◽  
Acid Sphingomyelinase ◽  
Pcr Analysis ◽  
Ceramide Synthase ◽  
Rt Pcr ◽  
Glucosylceramide Synthase ◽  
Expression Levels ◽  
Regulated Expression ◽  
Mrna Expression Levels

β-Sitosterol 3-O-d-glucoside (BSG) is known to act as an agonist by binding to estrogen receptors, and estrogen has been reported to enhance the activity of β-glucocerebrosidase, an epidermal ceramide metabolizing enzyme. In this study, we determined whether BSG up-regulates ceramide levels in the stratum corneum (SC) of a reconstructed human epidermal keratinization (RHEK) model. Treatment with BSG significantly increased the total ceramide content by 1.2-fold compared to that in the control in the SC of the RHEK model, accompanied by a significant increase of the ceramide species, Cer[EOS] by 2.1-fold compared to that in the control. RT-PCR analysis demonstrated that BSG significantly up-regulated the mRNA expression levels of serine palmitoyltransferase (SPT)2, ceramide synthase (CerS)3, glucosylceramide synthase (GCS) and acid sphingomyelinase by 1.41–1.89, 1.35–1.44, 1.19 and 2.06-fold, respectively, compared to that in the control in the RHEK model. Meanwhile, BSG significantly down-regulated the mRNA expression levels of sphingomyelin synthase (SMS)2 by 0.87–0.89-fold. RT-PCR analysis also demonstrated that BSG significantly up-regulated the mRNA expression levels of CerS3 and GCS by 1.19–1.55 and 1.20-fold, respectively, but not of SPT2 and significantly down-regulated that of SMS2 by 0.74-fold in HaCaT keratinocytes. Western blotting analysis revealed that BSG significantly increased the protein expression levels of CerS3 and GCS by 1.78 and 1.28–1.32-fold, respectively, compared to that in the control in HaCaT cells. These findings indicate that BSG stimulates ceramide synthesis via the up-regulated expression levels of CerS3 and GCS in the glucosylceramide pathway, which results in a significantly increased level of total ceramides in the SC accompanied by significantly increased levels of acylceramide species such as Cer[EOS].

Comparison of mRNA Expression Levels Determined with TAQ?Man and Competitive Template RT?PCR

ChemInform ◽  
2005 ◽  
Vol 36 (12) ◽  
Author(s):  
R. Mauritz ◽  
W. Schwabe ◽  
P. Haeusler ◽  
P. Noordhuis ◽  
K. Smid ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Rt Pcr ◽  
Expression Levels ◽  
Competitive Template ◽  
Mrna Expression Levels

Comparison of mRNA Expression Levels Determined with TAQ–Man and Competitive Template RT–PCR

2004 ◽  
Vol 23 (8-9) ◽  
pp. 1471-1474 ◽  
Author(s):  
R. Mauritz ◽  
W. Schwabe ◽  
P. Haeusler ◽  
P. Noordhuis ◽  
K. Smid ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Rt Pcr ◽  
Expression Levels ◽  
Competitive Template ◽  
Mrna Expression Levels

scholarly journals Interleukin-17 Can Induce Osteoarthritis in Rabbit Knee Joints Similar to Hulth’s Method

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Zili Wang ◽  
Chenhuang Zheng ◽  
Yunbin Zhong ◽  
Jinshen He ◽  
Xu Cao ◽  
...  
Keyword(s):  
Knee Joint ◽  
Mrna Expression ◽  
Histological Evaluation ◽  
Pcr Analysis ◽  
Control Group ◽  
Interleukin 17 ◽  
Expression Levels ◽  
X Ray ◽  
Rabbit Knee ◽  
Mrna Expression Levels

Interleukin-17 (IL-17) is closely related to osteoarthritis (OA), but animal studies that employ IL-17 to induce OA are currently lacking. Therefore, this study evaluated the effect of IL-17 in the rabbit knee joint. The right knees served as the control group. The left knees were divided randomly into 4 groups: a Hulth group and 3 IL-17 groups (1-ng, 10-ng, and 50-ng groups). OA was induced in the Hulth group using Hulth’s method. The IL-17 groups were injected with 1, 10, or 50 ng of IL-17 as indicated. Specimens were collected at 72 h, 1 week, 3 weeks, 6 weeks, and 12 weeks after surgery or the last injection. Subsequently, the following experiments were conducted: X-ray analysis, histological evaluation, and polymerase chain reaction (PCR) analysis of the mRNA expression levels of cartilage degeneration-related markers. At 12 weeks, like the Hulth group, the 10-ng and 50-ng IL-17 groups displayed typical manifestations of OA. The X-ray results, histological scores, and mRNA expression levels showed statistically significant differences between the control group and the 10-ng and 50-ng IL-17 groups. In sum, injecting 10 ng of IL-17 into the rabbit knee joint can induce OA similar to OA induced by Hulth’s method.

Targeted Reduction of Let-7a miRNA Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Jaira F. de Vasconcellos ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Megha Kaushal ◽  
Joshua M. Allwardt ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mirna Family ◽  
Fetal Hemoglobin ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Total Hemoglobin ◽  
Cd34 Cells ◽  
Mrna Expression Levels

Abstract MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

Developmental Changes of Growth Hormone Mrna Abundance in Pituitary Tissue of Jinhua and Landrace Gilts

2011 ◽  
Vol 343-344 ◽  
pp. 412-416
Author(s):  
Zhi Guo Miao ◽  
Guo Wang Li ◽  
Shi Zhu Wang ◽  
Xin Yao Chang ◽  
Hong Bing Xie ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Mrna Expression ◽  
Age Groups ◽  
Rt Pcr ◽  
Developmental Patterns ◽  
Expression Levels ◽  
Pituitary Tissue ◽  
Breed Differences ◽  
Gh Gene ◽  
Mrna Expression Levels

In this pepar we investigated the developmental patterns of expression of growth hormone (GH) gene in pituitary tissue in pigs of different breeds and their effects on the carcass fat contents. 3 Jinhua gilts and 3 Landrace gilts were sampled at 35, 80 and 125 days of age, respectively. Carcass fat contents were determined. Pituitary tissue was sampled and total RAN was extracted to determine GH mRNA expression levels by semi-quantitative RT-PCR. The results showed that the contents of carcass fat increased with growth and showed significant differences (P﹤0.05) between different age groups in the two breeds. Furthermore, carcass fat contents in Jinhua gilts were higher than that in Landrace gilts during growth (P﹤0.05). GH mRNA expression levels decreased with age and displayed breed differences. Jinhua gilts showed lower abundance of GH mRNA compared with Landrace gilts at 35, 80 and 125 days of age (P﹤0.05). In addition, GH mRNA expression level was negatively related to carcass fat content in Jinhua and Landrace gilts (r = -0.790 (P = 0.01), r = -0.755 (P = 0.02), respectively).

scholarly journals The role of CXCR3 and its ligands expression in Brucellar spondylitis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Hu ◽  
Xiaoqian Shang ◽  
Liang Wang ◽  
Jiahui Fan ◽  
Yue Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Inflammatory Infiltration ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Inflammatory Damage ◽  
Ifn Γ ◽  
Mrna Expression Levels

Abstract Aim Brucellar spondylitis (BS) is one of the most serious complications of brucellosis. CXCR3 is closely related to the severity of disease infection. This research aimed to study the degree of BS inflammatory damage through analyzing the expression levels of CXCR3 and its ligands (CXCL9 and CXCL10) in patients with BS. Methods A total of 29 BS patients and 15 healthy controls were enrolled. Real-Time PCR was used to detect the mRNA expression levels of IFN-γ, CXCR3, CXCL9 and CXCL10 in peripheral blood mononuclear cells (PBMCs) of BS patients and healthy controls. Hematoxylin-Eosin staining was used to show the pathological changes in BS lesion tissues. Immunohistochemistry staining was used to show the protein expression levels of Brucella-Ab, IFN-γ, CXCR3, CXCL9 and CXCL10 in BS lesion tissues. At the same time, ELISA was used to detect the serum levels of IFN-γ, CXCL9 CXCL10 and autoantibodies against CXCR3 in patients with BS. Results In lesion tissue of BS patients, it showed necrosis of cartilage, acute or chronic inflammatory infiltration. Brucella-Ab protein was abundantly expressed in close lesion tissue. And the protein expression levels of IFN-γ, CXCR3 and CXCL10 were highly expressed in close lesion tissue and serum of BS patients. At the same time, the mRNA expression levels of IFN-γ, CXCR3 and CXCL10 in PBMCs of BS patients were significantly higher than those in controls. Conclusion In our research, the expression levels of IFN-γ, CXCR3 and its ligands were significantly higher than those in controls. It suggested that high expression levels of IFN-γ, CXCR3 and its ligands indicated a serious inflammatory damage in patients with BS.

Quantitative RT-PCR analysis of MMP and TIMP MRNA expression in correlation to liver histology in patients with chronic hepatitis C

2000 ◽  
Vol 118 (4) ◽  
pp. A1469
Author(s):  
Dirk Michels ◽  
Christian I. Haberkorn ◽  
Burkhard Arndt ◽  
Michael P. Manns
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Mrna Expression ◽  
Chronic Hepatitis C ◽  
Liver Histology ◽  
Pcr Analysis ◽  
Rt Pcr
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